Loss of the Polyketide Synthase StlB Results in Stalk Cell Overproduction in Polysphondylium violaceum.

Major phenotypic innovations in social amoeba evolution occurred at the transition between the Polysphondylia and group 4 Dictyostelia, which comprise the model organism Dictyostelium discoideum, such as the formation of a new structure, the basal disk. Basal disk differentiation and robust stalk formation require the morphogen DIF-1, synthesized by the polyketide synthase StlB, the des-methyl-DIF-1 methyltransferase DmtA, and the chlorinase ChlA, which are conserved throughout Dictyostelia. To understand how the basal disk and other innovations evolved in group 4, we sequenced and annotated the Polysphondylium violaceum (Pvio) genome, performed cell type-specific transcriptomics to identify cell-type marker genes, and developed transformation and gene knock-out procedures for Pvio. We used the novel methods to delete the Pvio stlB gene. The Pvio stlB- mutants formed misshapen curly sorogens with thick and irregular stalks. As fruiting body formation continued, the upper stalks became more regular, but structures contained 40% less spores. The stlB- sorogens overexpressed a stalk gene and underexpressed a (pre)spore gene. Normal fruiting body formation and sporulation were restored in Pvio stlB- by including DIF-1 in the supporting agar. These data indicate that, although conserved, stlB and its product(s) acquired both a novel role in the group 4 Dictyostelia and a role opposite to that in its sister group.

(Auto)Biographical reflections on the contributions of William F. Loomis (1940-2016) to Dictyostelium biology.

William Farnsworth Loomis studied the social amoeba Dictyostelium discoideum for more than fifty years as a professor of biology at the University of California, San Diego, USA. This biographical reflection describes Dr. Loomis' major scientific contributions to the field within a career arc that spanned the early days of molecular biology up to the present day where the acquisition of high-dimensional datasets drive research. Dr. Loomis explored the genetic control of social amoeba development, delineated mechanisms of cell differentiation, and significantly advanced genetic and genomic technology for the field. The details of Dr. Loomis' multifaceted career are drawn from his published work, from an autobiographical essay that he wrote near the end of his career and from extensive conversations between him and the two authors, many of which took place on the deck of his beachfront home in Del Mar, California.

Microbiome management in the social amoeba Dictyostelium discoideum compared to humans.

Social amoebae and humans use common strategies to orchestrate their interactions with the bacteria in their respective environments and within their bodies. These strategies include the elimination of bacteria by phagocytosis, the establishment of mutualistic interactions, the elaboration of physical barriers, and the deployment of innate immune cells. Many of the molecular mechanisms that humans and social amoebae employ differ, but there are striking similarities that may inform studies in each organism. In this topical review we highlight the similarities and consider what we might learn by comparing these highly divergent species. We focus on recent work in Dictyostelium discoideum with hopes of stimulating work in this area and with the expectation that new mechanistic details uncovered in social amoebae-bacteria interactions will inform microbiome management in humans.

Social amoebae establish a protective interface with their bacterial associates by lectin agglutination.

Both animals and amoebae use phagocytosis and DNA-based extracellular traps as anti-bacterial defense mechanisms. Whether, like animals, amoebae also use tissue-level barriers to reduce direct contact with bacteria has remained unclear. We have explored this question in the social amoeba Dictyostelium discoideum, which forms plaques on lawns of bacteria that expand as amoebae divide and bacteria are consumed. We show that CadA, a cell adhesion protein that functions in D. discoideum development, is also a bacterial agglutinin that forms a protective interface at the plaque edge that limits exposure of vegetative amoebae to bacteria. This interface is important for amoebal survival when bacteria-to-amoebae ratios are high, optimizing amoebal feeding behavior, and protecting amoebae from oxidative stress. Lectins also control bacterial access to the gut epithelium of mammals to limit inflammatory processes; thus, this strategy of antibacterial defense is shared across a broad spectrum of eukaryotic taxa.

Cooperative predation in the social amoebae Dictyostelium discoideum.

The eukaryotic amoeba Dictyostelium discoideum is commonly used to study sociality. The amoebae cooperate during development, exhibiting altruism, cheating, and kin-discrimination, but growth while preying on bacteria has been considered asocial. Here we show that Dictyostelium are cooperative predators. Using mutants that grow poorly on Gram-negative bacteria but grow well on Gram-positive bacteria, we show that growth depends on cell-density and on prey type. We also found synergy, by showing that pairwise mixes of different mutants grow well on live Gram-negative bacteria. Moreover, wild-type amoebae produce diffusible factors that facilitate mutant growth and some mutants exploit the wild type in mixed cultures. Finding cooperative predation in D. discoideum should facilitate studies of this fascinating phenomenon, which has not been amenable to genetic analysis before.

Lectins modulate the microbiota of social amoebae.

The social amoeba Dictyostelium discoideum maintains a microbiome during multicellular development; bacteria are carried in migrating slugs and as endosymbionts within amoebae and spores. Bacterial carriage and endosymbiosis are induced by the secreted lectin discoidin I that binds bacteria, protects them from extracellular killing, and alters their retention within amoebae. This altered handling of bacteria also occurs with bacteria coated by plant lectins and leads to DNA transfer from bacteria to amoebae. Thus, lectins alter the cellular response of D. discoideum to bacteria to establish the amoebae's microbiome. Mammalian cells can also maintain intracellular bacteria when presented with bacteria coated with lectins, so heterologous lectins may induce endosymbiosis in animals. Our results suggest that endogenous or environmental lectins may influence microbiome homeostasis across eukaryotic phylogeny.

The polymorphic proteins TgrB1 and TgrC1 function as a ligand-receptor pair in Dictyostelium allorecognition.

Allorecognition is a key factor in Dictyostelium development and sociality. It is mediated by two polymorphic transmembrane proteins, TgrB1 and TgrC1, which contain extracellular immunoglobulin domains. TgrB1 and TgrC1 are necessary and sufficient for allorecognition, and they carry out separate albeit overlapping functions in development, but their mechanism of action is unknown. Here, we show that TgrB1 acts as a receptor with TgrC1 as its ligand in cooperative aggregation and differentiation. The proteins bind each other in a sequence-specific manner; TgrB1 exhibits a cell-autonomous function and TgrC1 acts non-cell-autonomously. The TgrB1 cytoplasmic tail is essential for its function and it becomes phosphorylated upon association with TgrC1. Dominant mutations in TgrB1 activate the receptor function and confer partial ligand independence. These roles in development and sociality suggest that allorecognition is crucial in the integration of individual cells into a coherent organism.

Social amoebae trap and kill bacteria by casting DNA nets.

Extracellular traps (ETs) from neutrophils are reticulated nets of DNA decorated with anti-microbial granules, and are capable of trapping and killing extracellular pathogens. Various phagocytes of mammals and invertebrates produce ETs, however, the evolutionary history of this DNA-based host defence strategy is unclear. Here we report that Sentinel (S) cells of the multicellular slug stage of the social amoeba Dictyostelium discoideum produce ETs upon stimulation with bacteria or lipopolysaccharide in a reactive oxygen species-dependent manner. The production of ETs by S cells requires a Toll/Interleukin-1 receptor domain-containing protein TirA and reactive oxygen species-generating NADPH oxidases. Disruption of these genes results in decreased clearance of bacterial infections. Our results demonstrate that D. discoideum is a powerful model organism to study the evolution and conservation of mechanisms of cell-intrinsic immunity, and suggest that the origin of DNA-based ETs as an innate immune defence predates the emergence of metazoans.

Gene Prioritization by Compressive Data Fusion and Chaining.

Data integration procedures combine heterogeneous data sets into predictive models, but they are limited to data explicitly related to the target object type, such as genes. Collage is a new data fusion approach to gene prioritization. It considers data sets of various association levels with the prediction task, utilizes collective matrix factorization to compress the data, and chaining to relate different object types contained in a data compendium. Collage prioritizes genes based on their similarity to several seed genes. We tested Collage by prioritizing bacterial response genes in Dictyostelium as a novel model system for prokaryote-eukaryote interactions. Using 4 seed genes and 14 data sets, only one of which was directly related to the bacterial response, Collage proposed 8 candidate genes that were readily validated as necessary for the response of Dictyostelium to Gram-negative bacteria. These findings establish Collage as a method for inferring biological knowledge from the integration of heterogeneous and coarsely related data sets.

Allorecognition, via TgrB1 and TgrC1, mediates the transition from unicellularity to multicellularity in the social amoeba Dictyostelium discoideum.

The social amoeba Dictyostelium discoideum integrates into a multicellular organism when individual starving cells aggregate and form a mound. The cells then integrate into defined tissues and develop into a fruiting body that consists of a stalk and spores. Aggregation is initially orchestrated by waves of extracellular cyclic adenosine monophosphate (cAMP), and previous theory suggested that cAMP and other field-wide diffusible signals mediate tissue integration and terminal differentiation as well. Cooperation between cells depends on an allorecognition system comprising the polymorphic adhesion proteins TgrB1 and TgrC1. Binding between compatible TgrB1 and TgrC1 variants ensures that non-matching cells segregate into distinct aggregates prior to terminal development. Here, we have embedded a small number of cells with incompatible allotypes within fields of developing cells with compatible allotypes. We found that compatibility of the allotype encoded by the tgrB1 and tgrC1 genes is required for tissue integration, as manifested in cell polarization, coordinated movement and differentiation into prestalk and prespore cells. Our results show that the molecules that mediate allorecognition in D. discoideum also control the integration of individual cells into a unified developing organism, and this acts as a gating step for multicellularity.

Genomic signatures of cooperation and conflict in the social amoeba.

Cooperative systems are susceptible to invasion by selfish individuals that profit from receiving the social benefits but fail to contribute. These so-called "cheaters" can have a fitness advantage in the laboratory, but it is unclear whether cheating provides an important selective advantage in nature. We used a population genomic approach to examine the history of genes involved in cheating behaviors in the social amoeba Dictyostelium discoideum, testing whether these genes experience rapid evolutionary change as a result of conflict over spore-stalk fate. Candidate genes and surrounding regions showed elevated polymorphism, unusual patterns of linkage disequilibrium, and lower levels of population differentiation, but they did not show greater between-species divergence. The signatures were most consistent with frequency-dependent selection acting to maintain multiple alleles, suggesting that conflict may lead to stalemate rather than an escalating arms race. Our results reveal the evolutionary dynamics of cooperation and cheating and underscore how sequence-based approaches can be used to elucidate the history of conflicts that are difficult to observe directly.

The ABC transporter, AbcB3, mediates cAMP export in D. discoideum development.

Extracellular cAMP functions as a primary ligand for cell surface cAMP receptors throughout Dictyostelium discoideum development, controlling chemotaxis and morphogenesis. The developmental consequences of cAMP signaling and the metabolism of cAMP have been studied in great detail, but it has been unclear how cells export cAMP across the plasma membrane. Here we show pharmacologically and genetically that ABC transporters mediate cAMP export. Using an evolutionary-developmental biology approach, we identified several candidate abc genes and characterized one of them, abcB3, in more detail. Genetic and biochemical evidence suggest that AbcB3 is a component of the cAMP export mechanism in D. discoideum development.

A novel human receptor involved in bitter tastant detection identified using Dictyostelium discoideum.

Detection of substances tasting bitter to humans occurs in diverse organisms including the social amoeba Dictyostelium discoideum. To establish a molecular mechanism for bitter tastant detection in Dictyostelium, we screened a mutant library for resistance to a commonly used bitter standard, phenylthiourea. This approach identified a G-protein-coupled receptor mutant, grlJ(-), which showed a significantly increased tolerance to phenylthiourea in growth, survival and movement. This mutant was not resistant to a structurally dissimilar potent bitter tastant, denatonium benzoate, suggesting it is not a target for at least one other bitter tastant. Analysis of the cell-signalling pathway involved in the detection of phenylthiourea showed dependence upon heterotrimeric G protein and phosphatidylinositol 3-kinase activity, suggesting that this signalling pathway is responsible for the cellular effects of phenylthiourea. This is further supported by a phenylthiourea-dependent block in the transient cAMP-induced production of phosphatidylinositol (3,4,5)-trisphosphate (PIP3) in wild-type but not grlJ(-) cells. Finally, we have identified an uncharacterized human protein γ-aminobutyric acid (GABA) type B receptor subunit 1 isoform with weak homology to GrlJ that restored grlJ(-) sensitivity to phenylthiourea in cell movement and PIP3 regulation. Our results thus identify a novel pathway for the detection of the standard bitter tastant phenylthiourea in Dictyostelium and implicate a poorly characterized human protein in phenylthiourea-dependent cell responses.

ABC transporters in Dictyostelium discoideum development.

ATP-binding cassette (ABC) transporters can translocate a broad spectrum of molecules across the cell membrane including physiological cargo and toxins. ABC transporters are known for the role they play in resistance towards anticancer agents in chemotherapy of cancer patients. There are 68 ABC transporters annotated in the genome of the social amoeba Dictyostelium discoideum. We have characterized more than half of these ABC transporters through a systematic study of mutations in their genes. We have analyzed morphological and transcriptional phenotypes for these mutants during growth and development and found that most of the mutants exhibited rather subtle phenotypes. A few of the genes may share physiological functions, as reflected in their transcriptional phenotypes. Since most of the abc-transporter mutants showed subtle morphological phenotypes, we utilized these transcriptional phenotypes to identify genes that are important for development by looking for transcripts whose abundance was unperturbed in most of the mutants. We found a set of 668 genes that includes many validated D. discoideum developmental genes. We have also found that abcG6 and abcG18 may have potential roles in intercellular signaling during terminal differentiation of spores and stalks.

Kin recognition protects cooperators against cheaters.

The evolution of sociality and altruism is enigmatic because cooperators are constantly threatened by cheaters who benefit from cooperation without incurring its full cost [1, 2]. Kin recognition is the ability to recognize and cooperate with genetically close relatives. It has also been proposed as a potential mechanism that limits cheating [3, 4], but there has been no direct experimental support for that possibility. Here we show that kin recognition protects cooperators against cheaters. The social amoebae Dictyostelium discoideum cooperate by forming multicellular aggregates that develop into fruiting bodies of viable spores and dead stalk cells. Cheaters preferentially differentiate into spores while their victims die as stalk cells in chimeric aggregates. We engineered syngeneic cheaters and victims that differed only in their kin-recognition genes, tgrB1 and tgrC1, and in a single cheater allele and found that the victims escaped exploitation by different types of nonkin cheaters. This protection depends on kin-recognition-mediated segregation because it is compromised when we disrupt strain segregation. These findings provide direct evidence for the role of kin recognition in cheater control and suggest a mechanism for the maintenance of stable cooperative systems.

Bacterial discrimination by dictyostelid amoebae reveals the complexity of ancient interspecies interactions.

BACKGROUND: Amoebae and bacteria interact within predator-prey and host-pathogen relationships, but the general response of amoeba to bacteria is not well understood. The amoeba Dictyostelium discoideum feeds on, and is colonized by, diverse bacterial species, including Gram-positive [Gram(+)] and Gram-negative [Gram(-)] bacteria, two major groups of bacteria that differ in structure and macromolecular composition. RESULTS: Transcriptional profiling of D. discoideum revealed sets of genes whose expression is enriched in amoebae interacting with different species of bacteria, including sets that appear specific to amoebae interacting with Gram(+) or with Gram(-) bacteria. In a genetic screen utilizing the growth of mutant amoebae on a variety of bacteria as a phenotypic readout, we identified amoebal genes that are only required for growth on Gram(+) bacteria, including one that encodes the cell-surface protein gp130, as well as several genes that are only required for growth on Gram(-) bacteria, including one that encodes a putative lysozyme, AlyL. These genes are required for parts of the transcriptional response of wild-type amoebae, and this allowed their classification into potential response pathways. CONCLUSIONS: We have defined genes that are critical for amoebal survival during feeding on Gram(+), or Gram(-), bacteria that we propose form part of a regulatory network that allows D. discoideum to elicit specific cellular responses to different species of bacteria in order to optimize survival.

A new social gene in Dictyostelium discoideum, chtB.

BACKGROUND: Competitive social interactions are ubiquitous in nature, but their genetic basis is difficult to determine. Much can be learned from single gene knockouts in a eukaryote microbe. The mutants can be competed with the parent to discern the social impact of that specific gene. Dictyostelium discoideum is a social amoeba that exhibits cooperative behavior in the construction of a multicellular fruiting body. It is a good model organism to study the genetic basis of cooperation since it has a sequenced genome and it is amenable to genetic manipulation. When two strains of D. discoideum are mixed, a cheater strain can exploit its social partner by differentiating more spore than its fair share relative to stalk cells. Cheater strains can be generated in the lab or found in the wild and genetic analyses have shown that cheating behavior can be achieved through many pathways. RESULTS: We have characterized the knockout mutant chtB, which was isolated from a screen for cheater mutants that were also able to form normal fruiting bodies on their own. When mixed in equal proportions with parental strain cells, chtB mutants contributed almost 60% of the total number of spores. To do so, chtB cells inhibit wild type cells from becoming spores, as indicated by counts and by the wild type cells' reduced expression of the prespore gene, cotB. We found no obvious fitness costs (morphology, doubling time in liquid medium, spore production, and germination efficiency) associated with the cheating ability of the chtB knockout. CONCLUSIONS: In this study we describe a new gene in D. discoideum, chtB, which when knocked out inhibits the parental strain from producing spores. Moreover, under lab conditions, we did not detect any fitness costs associated with this behavior.

Self-recognition in social amoebae is mediated by allelic pairs of tiger genes.

Free-living cells of the social amoebae Dictyostelium discoideum can aggregate and develop into multicellular fruiting bodies in which many die altruistically as they become stalk cells that support the surviving spores. Dictyostelium cells exhibit kin discrimination--a potential defense against cheaters, which sporulate without contributing to the stalk. Kin discrimination depends on strain relatedness, and the polymorphic genes tgrB1 and tgrC1 are potential components of that mechanism. Here, we demonstrate a direct role for these genes in kin discrimination. We show that a matching pair of tgrB1 and tgrC1 alleles is necessary and sufficient for attractive self-recognition, which is mediated by differential cell-cell adhesion. We propose that TgrB1 and TgrC1 proteins mediate this adhesion through direct binding. This system is a genetically tractable ancient model of eukaryotic self-recognition.

Comparative genomics of the social amoebae Dictyostelium discoideum and Dictyostelium purpureum.

BACKGROUND: The social amoebae (Dictyostelia) are a diverse group of Amoebozoa that achieve multicellularity by aggregation and undergo morphogenesis into fruiting bodies with terminally differentiated spores and stalk cells. There are four groups of dictyostelids, with the most derived being a group that contains the model species Dictyostelium discoideum. RESULTS: We have produced a draft genome sequence of another group dictyostelid, Dictyostelium purpureum, and compare it to the D. discoideum genome. The assembly (8.41 × coverage) comprises 799 scaffolds totaling 33.0 Mb, comparable to the D. discoideum genome size. Sequence comparisons suggest that these two dictyostelids shared a common ancestor approximately 400 million years ago. In spite of this divergence, most orthologs reside in small clusters of conserved synteny. Comparative analyses revealed a core set of orthologous genes that illuminate dictyostelid physiology, as well as differences in gene family content. Interesting patterns of gene conservation and divergence are also evident, suggesting function differences; some protein families, such as the histidine kinases, have undergone little functional change, whereas others, such as the polyketide synthases, have undergone extensive diversification. The abundant amino acid homopolymers encoded in both genomes are generally not found in homologous positions within proteins, so they are unlikely to derive from ancestral DNA triplet repeats. Genes involved in the social stage evolved more rapidly than others, consistent with either relaxed selection or accelerated evolution due to social conflict. CONCLUSIONS: The findings from this new genome sequence and comparative analysis shed light on the biology and evolution of the Dictyostelia.

Unconventional secretion of AcbA in Dictyostelium discoideum through a vesicular intermediate.

The acyl coenzyme A (CoA) binding protein AcbA is secreted unconventionally and processed into spore differentiation factor 2 (SDF-2), a peptide that coordinates sporulation in Dictyostelium discoideum. We report that AcbA is localized in vesicles that accumulate in the cortex of prespore cells just prior to sporulation. These vesicles are not observed after cells are stimulated to release AcbA but remain visible after stimulation in cells lacking the Golgi reassembly stacking protein (GRASP). Acyl-CoA binding is required for the inclusion of AcbA in these vesicles, and the secretion of AcbA requires N-ethylmaleimide-sensitive factor (NSF). About 1% of the total cellular AcbA can be purified within membrane-bound vesicles. The yield of vesicles decreases dramatically when purified from wild-type cells that were stimulated to release AcbA, whereas the yield from GRASP mutant cells was only modestly altered by stimulation. We suggest that these AcbA-containing vesicles are secretion intermediates and that GRASP functions at a late step leading to the docking/fusion of these vesicles at the cell surface.